Perspectives for Induced Pluripotent Stem Cell Technology: New Insights Into Human Physiology Involved in Somatic Mosaicism Naoki Nagata and Shinya Yamanaka Circ Res. 2014;114:505-510 doi: 10.1161/CIRCRESAHA.114.303043 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2014 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/114/3/505
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation Research is online at: http://circres.ahajournals.org//subscriptions/
Downloaded from http://circres.ahajournals.org/ at YORK UNIV SCOTT LIB on June 2, 2014
Review This article is in a thematic series on Recent Advances in iPS Cell Research, which includes the following articles: Steps Toward Safe Cell Therapy Using Induced Pluripotent Stem Cells (Circ Res. 2013;112:523–533) iPSCs in Cardiovascular Drug Discovery (Circ Res. 2013;112:534–548) Immunogenicity of Pluripotent Stem Cells and Their Derivatives (Circ Res. 2013;112:549–561) Progress in the Reprogramming of Somatic Cells (Circ Res. 2013;112:562–574) Direct Cardiac Reprogramming: From Developmental Biology to Cardiac Regeneration (Circ Res. 2013;113:915–921) Perspectives for iPS Cell Technology: New Insights Into Human Physiology Involved in Somatic Mosaicism Engineering Adolescence: Maturation of Human Pluripotent Stem Cell-Derived Cardiomyocytes iPS Cells for Post-myocardial Infarction Repair: Remarkable Opportunities and Challenges Shinya Yamanaka, Guest Editor
Perspectives for Induced Pluripotent Stem Cell Technology New Insights Into Human Physiology Involved in Somatic Mosaicism Naoki Nagata, Shinya Yamanaka
Abstract: Induced pluripotent stem cell technology makes in vitro reprogramming of somatic cells from individuals with various genetic backgrounds possible. By applying this technology, it is possible to produce pluripotent stem cells from biopsy samples of arbitrarily selected individuals with various genetic backgrounds and to subsequently maintain, expand, and stock these cells. From these induced pluripotent stem cells, target cells and tissues can be generated after certain differentiation processes. These target cells/tissues are expected to be useful in regenerative medicine, disease modeling, drug screening, toxicology testing, and proof-of-concept studies in drug development. Therefore, the number of publications concerning induced pluripotent stem cells has recently been increasing rapidly, demonstrating that this technology has begun to infiltrate many aspects of stem cell biology and medical applications. In this review, we discuss the perspectives of induced pluripotent stem cell technology for modeling human diseases. In particular, we focus on the cloning event occurring through the reprogramming process and its ability to let us analyze the development of complex disease-harboring somatic mosaicism. (Circ Res. 2014;114:505-510.) Key Words: induced pluripotent stem cells
I
nduced pluripotent stem (iPS) cell technology involves the generation of pluripotent stem cells from adult somatic cells by the exogenous expression of specific reprogramming factors1,2 (see Box). This technology opened the door for us to access a robust platform demonstrating in vitro cellular reprogramming from certain somatic cells to pluripotent status. Recently, Cahan and Daley8 conducted a bibliometric analysis of the pluripotent stem cells (including iPS cells, embryonic stem [ES] cells, and embryonic carcinoma cells) generated since 2010. They found that the number of publications concerning ES cells has decreased, whereas the number of publications
■
mosaicism
concerning the applications of pluripotent stem cells has increased. They pointed out that it is the increase in the studies of iPS cells that brought about this change in the research trend, because it gave scientists access to pluripotent stem cells with robust and reproducible technology. As Cahan and Daley8 have shown, iPS cells quickly became recognized as an innovative research tool for the study of biology and medicine. In this review, we focus on iPS cell technology, with a special emphasis on its cloning event, which is essential for the reprogramming process, and discuss perspectives related to the creation of in vitro models of human physiological conditions, including complex diseases.
Original received November 15, 2013; revision received December 27, 2013; accepted January 6, 2014. In December 2013, the average time from submission to first decision for all original research papers submitted to Circulation Research was 11.66 days. From the Department of Reprogramming Science, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan (N.N., S.Y.); and Gladstone Institute of Cardiovascular Disease, San Francisco, CA (S.Y.). Correspondence to Shinya Yamanaka, MD, PhD, Center for iPS cell Research and Application, Kyoto University, Kyoto 606-8507, Japan. E-mail [email protected] © 2014 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.114.303043
Downloaded from http://circres.ahajournals.org/ 505at YORK UNIV SCOTT LIB on June 2, 2014
506 Circulation Research January 31, 2014
Nonstandard Abbreviations and Acronyms CINCA DS ES iPS
chronic infantile neurologic cutaneous and articular Down syndrome embryonic stem induced pluripotent stem
Discovery of Induced Pluripotent Stem Cells Induced pluripotent stem (iPS) cells are generated from mouse cells by the ectopic expression of key transcription factors (Oct3/4, Sox2, c-Myc, Klf4).1 The iPS cells were indistinguishable from embryonic stem cells in terms of their morphology, gene expression, self-renewal capacity, and pluripotency. Therefore, under this process, the somatic memory of the subject cells was reprogrammed to the stage of blastocyst inner cells. Further experiments confirmed the contribution of iPS cells to mammalian (mouse) ontogenesis. The mouse iPS cells have been confirmed to have a chimeric contribution following blastocyst injection and have also been proven to have competency for the germ line.3–5 Subsequently, human iPS cells were established by 3 laboratories at almost the same time using the same approaches.2,6,7 This shows that the iPS cell technology was already highly reproducible and accessible to several laboratories and that these cells can be readily used to perform experiments in molecular biology and mammalian cell culture. These iPS cells met the criteria proposed for the definition of human embryonic stem cells, with the exception that the iPS cells are not derived from embryos.
iPS Cells for In Vitro Modeling of Native Physiological Conditions Immortalized cells and primary cell lines have been the candidate cells used for creating human physiological models thus far. However, both of these types of cells are associated with serious defects when used for such purposes. Although immortalized cell lines can be maintained as homogenous cell populations at low cost, they lack important aspects of the native functions of cells. Likewise, although primary cell lines can provide fully differentiated cells that have a close approximation of the native function, the collection of all cell types is not possible because of the level of invasion required to collect them, and the limited proliferative activity of primary cells can make reproducing experiments difficult.9 On the contrary, human-derived iPS and ES cell lines can overcome these disadvantages. After the expansion of these pluripotent cell lines, a sufficient number of the desired types of cells can be prepared by using appropriate differentiation protocols, that is, these technologies can provide an opportunity to maintain specific somatic cells on a large scale and in a renewable way. However, there are still some limitations that need to be overcome. For example, it has been pointed out that cardiomyocytes differentiated from iPS cells using the current differentiation protocols yield relatively immature cells.10–12 Therefore, further improvement of the differentiation and
maturation protocols will be needed to more accurately recapitulate the phenotype in iPS cell–derived differentiated cells. Human ES cells carrying mutation for some genetic disorders have been reported (eg, Huntington disease, cystic fibrosis, myotonic dystrophy type 1, and Fragile X syndrome).13,14 However, there are certain ethical concerns associated with developing ES cells obtained from in vitro fertilization clinics for the study of genetic diseases.15 In contrast, such ethical issues are overcome with the use of disease-specific iPS cell lines, because they can be generated from somatic cells such as fibroblasts or peripheral blood cells, which can be collected by minimally invasive procedures and do not require the destruction of a human embryo.
iPS Cells From Arbitrarily Selected Individuals Another advantage of using iPS cells in medical research is that this technology makes it possible to obtain human pluripotent stem cells from arbitrarily selected individuals with various genetic backgrounds, including patients with various diseases. In 2008, the first patient-specific iPS cells were generated.16,17 Since then, abundant studies of disease models that represent the human condition with high fidelity have been reported,18,19 particularly those aiming to investigate diseases that are caused by a single gene mutation. In most of the studies, iPS cell lines from unaffected or healthy donors were used as controls. However, recent genome-wide association studies using a set of controls derived from different individuals showed significant experimental noise attributable to genomic variations. Moreover, these controls cannot really be defined as healthy, because each person carries 50 to 100 diseaseassociated genetic variations.20 One way to address these issues is to obtain isogenic controls by correcting mutated genes in patient-specific iPS cell lines.21–27 The recent progresses in genetic engineering technologies, such as Transcription Activator-Like Effector Nuclease (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs), will facilitate this strategy.27,28 Somatic mutations can give rise to a broad range of diseases, including cancer, and noncancerous mosaic diseases.18 Recently, heterogeneous patient-specific iPS cell lines have been generated from the somatic cells of patients with various mosaic diseases, such as Down syndrome (DS),29–35 Fragile X syndrome,36 Rett syndrome,37–42 Fanconi anemia,43 and chronic infantile neurologic cutaneous and articular (CINCA) syndrome.44 Even though there are some limitations associated with the kinds of diseases that can be modeled, using iPS cells derived from patients with somatic mosaicism has an advantage in that it allows analyses with isogenic controls. In these cases, both mutant and wild-type iPS cell clones can be generated from the same patients. Each pair of clones theoretically has the same genetic background, except for the altered chromosome or mutated gene(s) and should serve as an ideal pair of mutant and isogenic control clones, thereby allowing for highly reliable studies of the impact of the mutation. It should be mentioned that we could not completely exclude the introduction of new mutations during the induction of iPS cells and subsequent cultivation. The recently improved next-generation DNA sequencing and bioinformatics techniques should be useful in detecting such acquired mutations and increasing the quality of experiments.
Downloaded from http://circres.ahajournals.org/ at YORK UNIV SCOTT LIB on June 2, 2014
Nagata and Yamanaka Perspectives for iPS Cell Technology 507 The following examples of patient-specific iPS cells generated from mosaic cases of DS and CINCA syndrome illustrate the advantage of using iPS cell technology for disease modeling.
Somatic Mosaicism in DS DS is the most frequent form of mental retardation and is caused by autosomal trisomy of all or a critical portion of chromosome 21. Patients with DS are reported to present with multiple disorders (eg, congenital heart defects, particularly atrioventricular septal defect, leukemia, and early- onset Alzheimer disease [OMIM 190685]).45 A cytogenetic study showed that, in the United States, most infants with DS (≈95%) had full trisomy 21; in ≈3% of patients, one copy was translocated to another acrocentric chromosome, and in ≈2% of cases, live-born trisomy 21 individuals were recognized to have mosaicism for a trisomic cell line.46 Mosaicism results from the abnormal division of some cells after fertilization, with some of the cells having 47 chromosomes and the others being normal. In each of these conditions, the outcome of this disorder is determined by the particular chromosome being duplicated, as well as by the proportion of cells in the body carrying the abnormality.18 Papavassiliou et al47 observed a correlation between the frequency of trisomic cells and the patient phenotype. They detected a significant inverse correlation between the frequency of trisomic cells and IQ scores in individuals with mosaic trisomy 21. Mosaic trisomies have also been reported on other chromosomes, such as chromosomes 1, 8, 9, 13, 16, 17, 18, and 22 (as reviewed by Poduri et al18). Many attempts have been made to model DS to investigate the detailed pathophysiology of the condition. Generally, biopsy samples of brains are not available from patients, and thus, mouse models are currently the primary tools used to study the pathogenesis of DS. However, brain development differs in mice and humans.48 Although some human ES cells with trisomy 21 have been generated,49 the establishment of human ES cells from patients with DS remains ethically challenging.
Capturing Trisomic and Disomic Cells From Mosaic Patients Using the iPS Cell Technology: DS as an Example DS/trisomy 21 patient-specific iPS cell lines have been generated from various cases, including mosaic patients.17,29–35
DS Patient-derived iPS cell lines
DS-Patient / Mosaicism Biopsy sample
Reprogramming
Cloning
Disomic cell
Trisomic cell Transcription factors
Figure. Generation of induced pluripotent stem (iPS) cell lines from patient with mosaic Down syndrome (DS).
Recently, 2 groups reported the generation of iPS cell lines from fibroblasts that were derived from patients with DS with mosaicism.34,48 They confirmed that the trisomy 21 karyotype was present in the DS iPS cells; however, they also captured iPS cell lines from the patient’s fibroblasts, which were found to possess a disomic karyotype. This seemed to be the result of the reprogramming of euploid cells from the mosaic donor’s fibroblast population. DNA profiling of the corresponding cell lines revealed that these disomic and trisomic cell lines actually came from the same donor. The patient’s disomic cell line provides an ideal experimental control, because it can be used cancel the noise in the genetic background that is often observed using so-called “healthy” individuals as controls. This system presents a powerful method for analyzing multiple genotype–phenotype associations in complex diseases that harbor multiple candidate mutations, such as chromosomal aberrations (Figure).
Somatic Mosaicism in CINCA Syndrome Beside DS, patient-specific iPS cell lines have also been generated from the somatic cells of patients with autosomal mosaic point mutations. For example, CINCA syndrome44 is a dominantly inherited autoinflammatory disease characterized by cutaneous symptoms, central nervous system involvement, and arthropathy (OMIM 607115). Saito et al50 described the first case of a patient with somatic mosaicism in their NLRP3 (also known as CIAS1) gene mutation. It has been observed that 30% to 40% of all patients have mutations in NLRP3 in only a small number (≈10%) of somatic cells,50,51 despite the fact that nearly half of all patients with CINCA syndrome carry heterozygous gain-of-function mutations of the gene.9,52 Recently, Tanaka et al44 generated both NLP3 mutant and nonmutant iPS cell lines from patients with CINCA syndrome with somatic mosaicism and described their differentiation into macrophages. In this case, they succeeded in recapitulating the disease-relevant phenotype using only mutant macrophages derived from iPS cells, demonstrating that NLRP3-mutant macrophages are responsible for the pathogenesis of mosaic CINCA syndrome. Moreover, they succeeded in developing a drug screening system with the macrophages derived from iPS cells and illustrated the usefulness of iPS cell technology as a platform for drug discovery.
A New Approach to Studying Somatic Mutations Using the iPS Cell Technology Recently, somatic mosaicism has been reported in a variety of tissues from healthy individuals, suggesting that it has physiological functions.29 In such cases, iPS cell technology can provide a universally applicable strategy to capture these somatic mosaicisms in human populations, allowing their impact to be evaluated. Evidence from recent studies using single-nucleotide polymorphism microarrays or the next-generation sequencing technology demonstrates that a certain proportion of mutations that are detected in iPS cells come from the heterogeneous donor cell population, attributable to somatic mosaicism.53–55 The cells that carry pre-existing genetic alterations, such as singlenucleotide variants and copy number variations, are captured by the cloning event through the reprogramming process.
Downloaded from http://circres.ahajournals.org/ at YORK UNIV SCOTT LIB on June 2, 2014
508 Circulation Research January 31, 2014 These single-nucleotide variants and copy number variations in a minor population of somatic cells have to date been overlooked because of the difficulty in detecting them. However, applying iPS cell technology allows for improved detection of such low-level mosaicism.
environmental/aging association successfully at the molecular level by applying iPS cell technology. Moreover, iPS cell technology holds the potential to lead to new insights into the human physiology associated with somatic mosaicism.
Somatic Mosaicisms in Cardiovascular Diseases
We thank Drs Yoshinori Yoshida and Kazutoshi Takahashi for their valuable scientific discussions. We also thank Rie Kato and Yoko Miyake for their valuable administrative support.
Systematic analyses of Leipzig heart collections have shown somatic mutations in several transcription factor genes (NKX2-5, TBX5, GATA4, and HEY2) associated with complex congenital heart diseases, including atrial septal, ventricular, and atrioventricular septal defects.56–61 In these cases, each mutation was detected in the affected heart tissues, but not in the normal heart tissues, of the same patients. In this situation, mutant cardiomyocytes can be provided by gene editing of wild-type iPS cell lines. Another set of examples of mosaicism was found in somatic mutations associated with arrhythmia.62–64 Mosaicism in connexin 40 and connexin 43 is associated with atrial fibrillation.63,64 Interestingly, subcloning analysis estimated allelic frequencies of mutant alleles at 20% to 34% within the patient’s cardiac tissue specimens; however, these mutations were not detected in lymphocytes. Each mutant connexin- expressing cell was observed loss of contribution to gap junction formations, as well as electrophysiological functions. To further investigate the pathophysiology of those atrial fibrillations with somatic mosaicism of connexins, modeling of in vitro cardiac syncytia in which each cardiomyocyte is individually expressing either wild-type or mutant connexins provides a powerful tool. Recently, Kadota et al65 created cardiomyocyte sheets from human iPS/ES cells. They demonstrated that these sheets are capable of generating re-entrant arrhythmia models when stimulated with high-frequency electric pulses and subsequently showed their usefulness for screening and testing drugs with antiarrhythmic potential. Although further technological improvements are needed, this approach can provide a more precise in vitro micro–re-entrant model for the mosaic cardiac syncytium. In this case, we may choose to obtain mutated cardiac cells by gene-editing technology based on unaffected/well-characterized iPS cells because to date mutant connexin 40–expressing and connexin 43–expressing cells have only been detected in patient cardiac tissues, which are difficult to obtain by biopsy.
Concluding Remarks Somatic variants are known to be potentially responsible for various diseases. However, the extent of somatic variation may have been markedly underestimated.66 As we discussed above, iPS cell technology allows us to approach human biology with higher resolution at the cellular level through the cell cloning process, as part of the reprogramming process. This approach holds great promise for studies on the native human physiological processes, as well as the development and pathology of human diseases. For example, in patients with trisomy 21, acquired mosaicism was observed in adult patients.67 Although there are still several technical hurdles that have to be overcome, we may be able to analyze such a disease/
Acknowledgments
Sources of Funding This work was supported by a grant from the Funding Program for World-Leading Innovative Research and Development on Science and Technology (FIRST Program) of the Japanese Society for the Promotion of Science (JSPS) and by a grant from Research Center Network for Realization of Regenerative Medicine of the Japan Science and Technology Agency (JST).
Disclosures S. Yamanaka is a member without salary of the scientific advisory boards of iPierian, iPS Academia Japan, Megakaryon Corporation, and HEALIOS K. K. Japan. The other author reports no conflicts.
References 1. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663–676. 2. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131:861–872. 3. Maherali N, Sridharan R, Xie W, Utikal J, Eminli S, Arnold K, Stadtfeld M, Yachechko R, Tchieu J, Jaenisch R, Plath K, Hochedlinger K. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell. 2007;1:55–70. 4. Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature. 2007;448:313–317. 5. Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, Hochedlinger K, Bernstein BE, Jaenisch R. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature. 2007;448:318–324. 6. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007;318:1917–1920. 7. Park IH, Zhao R, West JA, Yabuuchi A, Huo H, Ince TA, Lerou PH, Lensch MW, Daley GQ. Reprogramming of human somatic cells to pluripotency with defined factors. Nature. 2008;451:141–146. 8. Cahan P, Daley GQ. Origins and implications of pluripotent stem cell variability and heterogeneity. Nat Rev Mol Cell Biol. 2013;14:357–368. 9. Ebert AD, Shelley BC, Hurley AM, Onorati M, Castiglioni V, Patitucci TN, Svendsen SP, Mattis VB, McGivern JV, Schwab AJ, Sareen D, Kim HW, Cattaneo E, Svendsen CN. EZ spheres: a stable and expandable culture system for the generation of pre-rosette multipotent stem cells from human ESCs and iPSCs. Stem Cell Res. 2013;10:417–427. 10. Jung CB, Moretti A, Mederos y Schnitzler M, et al. Dantrolene rescues arrhythmogenic RYR2 defect in a patient-specific stem cell model of catecholaminergic polymorphic ventricular tachycardia. EMBO Mol Med. 2012;4:180–191. 11. Dirschinger RJ, Goedel A, Moretti A, Laugwitz KL, Sinnecker D. Recapitulating long-QT syndrome using induced pluripotent stem cell technology. Pediatr Cardiol. 2012;33:950–958. 12. Friedrichs S, Malan D, Sasse P. Modeling long QT syndromes using induced pluripotent stem cells: current progress and future challenges. Trends Cardiovasc Med. 2013;23:91–98. 13. Mateizel I, De Temmerman N, Ullmann U, Cauffman G, Sermon K, Van de Velde H, De Rycke M, Degreef E, Devroey P, Liebaers I, Van Steirteghem A. Derivation of human embryonic stem cell lines from embryos obtained after IVF and after PGD for monogenic disorders. Hum Reprod. 2006;21:503–511. 14. Eiges R, Urbach A, Malcov M, Frumkin T, Schwartz T, Amit A, Yaron Y, Eden A, Yanuka O, Benvenisty N, Ben-Yosef D. Developmental
Downloaded from http://circres.ahajournals.org/ at YORK UNIV SCOTT LIB on June 2, 2014
Nagata and Yamanaka Perspectives for iPS Cell Technology 509 study of fragile X syndrome using human embryonic stem cells derived from preimplantation genetically diagnosed embryos. Cell Stem Cell. 2007;1:568–577. 15. Pomp O, Colman A. Disease modelling using induced pluripotent stem cells: status and prospects. Bioessays. 2013;35:271–280. 16. Dimos JT, Rodolfa KT, Niakan KK, Weisenthal LM, Mitsumoto H, Chung W, Croft GF, Saphier G, Leibel R, Goland R, Wichterle H, Henderson CE, Eggan K. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science. 2008;321:1218–1221. 17. Park IH, Arora N, Huo H, Maherali N, Ahfeldt T, Shimamura A, Lensch MW, Cowan C, Hochedlinger K, Daley GQ. Disease-specific induced pluripotent stem cells. Cell. 2008;134:877–886. 18. Poduri A, Evrony GD, Cai X, Walsh CA. Somatic mutation, genomic variation, and neurological disease. Science. 2013;341:1237758. 19. Trounson A, Shepard KA, DeWitt ND. Human disease modeling with induced pluripotent stem cells. Curr Opin Genet Dev. 2012;22:509–516. 20. Abecasis GR, Altshuler D, Auton A, Brooks LD, Durbin RM, Gibbs RA, Hurles ME, McVean GA; 1000 Genomes Project Consortium. A map of human genome variation from population-scale sequencing. Nature. 2010;467:1061–1073. 21. Kazuki Y, Hiratsuka M, Takiguchi M, et al. Complete genetic cor rection of ips cells from Duchenne muscular dystrophy. Mol Ther. 2010;18:386–393. 22. Howden SE, Gore A, Li Z, et al. Genetic correction and analysis of induced pluripotent stem cells from a patient with gyrate atrophy. Proc Natl Acad Sci U S A. 2011;108:6537–6542. 23. Liu GH, Suzuki K, Qu J, et al. Targeted gene correction of laminopathyassociated LMNA mutations in patient-specific iPSCs. Cell Stem Cell. 2011;8:688–694. 24. Tolar J, Park IH, Xia L, Lees CJ, Peacock B, Webber B, McElmurry RT, Eide CR, Orchard PJ, Kyba M, Osborn MJ, Lund TC, Wagner JE, Daley GQ, Blazar BR. Hematopoietic differentiation of induced pluripotent stem cells from patients with mucopolysaccharidosis type I (Hurler syndrome). Blood. 2011;117:839–847. 25. Bellin M, Casini S, Davis RP, D’Aniello C, Haas J, Ward-van Oostwaard D, Tertoolen LG, Jung CB, Elliott DA, Welling A, Laugwitz KL, Moretti A, Mummery CL. Isogenic human pluripotent stem cell pairs reveal the role of a KCNH2 mutation in long-QT syndrome. EMBO J. 2013;32:3161–3175. 26. Fattahi F, Asgari S, Pournasr B, Seifinejad A, Totonchi M, Taei A, Aghdami N, Salekdeh GH, Baharvand H. Disease-corrected hepatocytelike cells from familial hypercholesterolemia-induced pluripotent stem cells. Mol Biotechnol. 2013;54:863–873. 27. Yusa K, Rashid ST, Strick-Marchand H, et al. Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells. Nature. 2011;478:391–394. 28. Merkle FT, Eggan K. Modeling human disease with pluripotent stem cells: from genome association to function. Cell Stem Cell. 2013;12:656–668. 29. Shi Y, Kirwan P, Smith J, MacLean G, Orkin SH, Livesey FJ. A human stem cell model of early Alzheimer’s disease pathology in Down syndrome. Sci Transl Med. 2012;4:124ra29. 30. Mou X, Wu Y, Cao H, Meng Q, Wang Q, Sun C, Hu S, Ma Y, Zhang H. Generation of disease-specific induced pluripotent stem cells from patients with different karyotypes of Down syndrome. Stem Cell Res Ther. 2012;3:14. 31. Li LB, Chang KH, Wang PR, Hirata RK, Papayannopoulou T, Russell DW. Trisomy correction in Down syndrome induced pluripotent stem cells. Cell Stem Cell. 2012;11:615–619. 32. Biesecker LG, Spinner NB. A genomic view of mosaicism and human disease. Nat Rev Genet. 2013;14:307–320. 33. Maclean GA, Menne TF, Guo G, Sanchez DJ, Park IH, Daley GQ, Orkin SH. Altered hematopoiesis in trisomy 21 as revealed through in vitro differentiation of isogenic human pluripotent cells. Proc Natl Acad Sci U S A. 2012;109:17567–17572. 34. Weick JP, Held DL, Bonadurer GF 3rd, et al. Deficits in human trisomy 21 iPSCs and neurons. Proc Natl Acad Sci U S A. 2013;110:9962–9967. 35. Lu HE, Yang YC, Chen SM, Su HL, Huang PC, Tsai MS, Wang TH, Tseng CP, Hwang SM. Modeling neurogenesis impairment in Down syndrome with induced pluripotent stem cells from Trisomy 21 amniotic fluid cells. Exp Cell Res. 2013;319:498–505. 36. Sheridan SD, Theriault KM, Reis SA, Zhou F, Madison JM, Daheron L, Loring JF, Haggarty SJ. Epigenetic characterization of the FMR1 gene and aberrant neurodevelopment in human induced pluripotent stem cell models of fragile X syndrome. PLoS One. 2011;6:e26203.
37. Marchetto MC, Carromeu C, Acab A, Yu D, Yeo GW, Mu Y, Chen G, Gage FH, Muotri AR. A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell. 2010;143:527–539. 38. Amenduni M, De Filippis R, Cheung AY, Disciglio V, Epistolato MC, Ariani F, Mari F, Mencarelli MA, Hayek Y, Renieri A, Ellis J, Meloni I. iPS cells to model CDKL5-related disorders. Eur J Hum Genet. 2011;19:1246–1255. 39. Ananiev G, Williams EC, Li H, Chang Q. Isogenic pairs of wild type and mutant induced pluripotent stem cell (iPSC) lines from Rett syndrome patients as in vitro disease model. PLoS One. 2011;6:e25255. 40. Cheung AY, Horvath LM, Grafodatskaya D, Pasceri P, Weksberg R, Hotta A, Carrel L, Ellis J. Isolation of MECP2-null Rett Syndrome patient hiPS cells and isogenic controls through X-chromosome inactivation. Hum Mol Genet. 2011;20:2103–2115. 41. Kim KY, Hysolli E, Park IH. Neuronal maturation defect in induced pluripotent stem cells from patients with Rett syndrome. Proc Natl Acad Sci U S A. 2011;108:14169–14174. 42. Pomp O, Dreesen O, Leong DF, Meller-Pomp O, Tan TT, Zhou F, Colman A. Unexpected X chromosome skewing during culture and reprogramming of human somatic cells can be alleviated by exogenous telomerase. Cell Stem Cell. 2011;9:156–165. 43. Müller LU, Schlaeger TM, DeVine AL, Williams DA. Induced pluripotent stem cells as a tool for gaining new insights into Fanconi anemia. Cell Cycle. 2012;11:2985–2990. 44. Tanaka T, Takahashi K, Yamane M, et al. Induced pluripotent stem cells from CINCA syndrome patients as a model for dissecting somatic mosaicism and drug discovery. Blood. 2012;120:1299–1308. 45. Mégarbané A, Ravel A, Mircher C, Sturtz F, Grattau Y, Rethoré MO, Delabar JM, Mobley WC. The 50th anniversary of the discovery of trisomy 21: the past, present, and future of research and treatment of Down syndrome. Genet Med. 2009;11:611–616. 46. Shin M, Siffel C, Correa A. Survival of children with mosaic Down syndrome. Am J Med Genet A. 2010;152A:800–801. 47. Papavassiliou P, York TP, Gursoy N, Hill G, Nicely LV, Sundaram U, McClain A, Aggen SH, Eaves L, Riley B, Jackson-Cook C. The phenotype of persons having mosaicism for trisomy 21/Down syndrome reflects the percentage of trisomic cells present in different tissues. Am J Med Genet A. 2009;149A:573–583. 48. Briggs JA, Sun J, Shepherd J, Ovchinnikov DA, Chung TL, Nayler SP, Kao LP, Morrow CA, Thakar NY, Soo SY, Peura T, Grimmond S, Wolvetang EJ. Integration-free induced pluripotent stem cells model genetic and neural developmental features of down syndrome etiology. Stem Cells. 2013;31:467–478. 49. Biancotti JC, Narwani K, Buehler N, Mandefro B, Golan-Lev T, Yanuka O, Clark A, Hill D, Benvenisty N, Lavon N. Human embryonic stem cells as models for aneuploid chromosomal syndromes. Stem Cells. 2010;28:1530–1540. 50. Saito M, Fujisawa A, Nishikomori R, Kambe N, Nakata-Hizume M, Yoshimoto M, Ohmori K, Okafuji I, Yoshioka T, Kusunoki T, Miyachi Y, Heike T, Nakahata T. Somatic mosaicism of CIAS1 in a patient with chronic infantile neurologic, cutaneous, articular syndrome. Arthritis Rheum. 2005;52:3579–3585. 51. Tanaka N, Izawa K, Saito MK, et al. High incidence of NLRP3 somatic mosaicism in patients with chronic infantile neurologic, cutaneous, articular syndrome: results of an International Multicenter Collaborative Study. Arthritis Rheum. 2011;63:3625–3632. 52. Feldmann J, Prieur AM, Quartier P, Berquin P, Certain S, Cortis E, TeillacHamel D, Fischer A, de Saint Basile G. Chronic infantile neurological cutaneous and articular syndrome is caused by mutations in CIAS1, a gene highly expressed in polymorphonuclear cells and chondrocytes. Am J Hum Genet. 2002;71:198–203. 53. Abyzov A, Mariani J, Palejev D, et al. Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells. Nature. 2012;492:438–442. 54. Young MA, Larson DE, Sun CW, et al. Background mutations in parental cells account for most of the genetic heterogeneity of induced pluripotent stem cells. Cell Stem Cell. 2012;10:570–582. 55. Chou ST, Byrska-Bishop M, Tober JM, Yao Y, Vandorn D, Opalinska JB, Mills JA, Choi JK, Speck NA, Gadue P, Hardison RC, Nemiroff RL, French DL, Weiss MJ. Trisomy 21-associated defects in human primitive hematopoiesis revealed through induced pluripotent stem cells. Proc Natl Acad Sci U S A. 2012;109:17573–17578. 56. Reamon-Buettner SM, Borlak J. Somatic NKX2-5 mutations as a novel mechanism of disease in complex congenital heart disease. J Med Genet. 2004;41:684–690.
Downloaded from http://circres.ahajournals.org/ at YORK UNIV SCOTT LIB on June 2, 2014
510 Circulation Research January 31, 2014 57. Reamon-Buettner SM, Hecker H, Spanel-Borowski K, Craatz S, Kuenzel E, Borlak J. Novel NKX2-5 mutations in diseased heart tissues of patients with cardiac malformations. Am J Pathol. 2004;164: 2117–2125. 58. Reamon-Buettner SM, Borlak J. TBX5 mutations in non-Holt-Oram syndrome (HOS) malformed hearts. Hum Mutat. 2004;24:104. 59. Reamon-Buettner SM, Borlak J. GATA4 zinc finger mutations as a molecular rationale for septation defects of the human heart. J Med Genet. 2005;42:e32. 60. Reamon-Buettner SM, Borlak J. HEY2 mutations in malformed hearts. Hum Mutat. 2006;27:118. 61. Weismann CG, Gelb BD. The genetics of congenital heart disease: a review of recent developments. Curr Opin Cardiol. 2007;22:200–206. 62. Lerman BB, Dong B, Stein KM, Markowitz SM, Linden J, Catanzaro DF. Right ventricular outflow tract tachycardia due to a somatic cell mutation in G protein subunitalphai2. J Clin Invest. 1998;101:2862–2868.
63. Gollob MH, Jones DL, Krahn AD, et al. Somatic mutations in the connexin 40 gene (GJA5) in atrial fibrillation. N Engl J Med. 2006;354:2677–2688. 64. Thibodeau IL, Xu J, Li Q, Liu G, Lam K, Veinot JP, Birnie DH, Jones DL, Krahn AD, Lemery R, Nicholson BJ, Gollob MH. Paradigm of genetic mosaicism and lone atrial fibrillation: physiological characterization of a connexin 43-deletion mutant identified from atrial tissue. Circulation. 2010;122:236–244. 65. Kadota S, Minami I, Morone N, Heuser JE, Agladze K, Nakatsuji N. Development of a reentrant arrhythmia model in human pluripotent stem cell-derived cardiac cell sheets. Eur Heart J. 2013;34:1147–1156. 66. Erickson RP. Somatic gene mutation and human disease other than cancer: an update. Mutat Res. 2010;705:96–106. 67. Jenkins EC, Schupf N, Genovese M, Ye LL, Kapell D, Canto B, Harris M, Devenny D, Lee JH, Brown WT. Increased low-level chromosome 21 mosaicism in older individuals with Down syndrome. Am J Med Genet. 1997;68:147–151.
Downloaded from http://circres.ahajournals.org/ at YORK UNIV SCOTT LIB on June 2, 2014
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/114/3/505
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation Research is online at: http://circres.ahajournals.org//subscriptions/
Downloaded from http://circres.ahajournals.org/ at YORK UNIV SCOTT LIB on June 2, 2014
Review This article is in a thematic series on Recent Advances in iPS Cell Research, which includes the following articles: Steps Toward Safe Cell Therapy Using Induced Pluripotent Stem Cells (Circ Res. 2013;112:523–533) iPSCs in Cardiovascular Drug Discovery (Circ Res. 2013;112:534–548) Immunogenicity of Pluripotent Stem Cells and Their Derivatives (Circ Res. 2013;112:549–561) Progress in the Reprogramming of Somatic Cells (Circ Res. 2013;112:562–574) Direct Cardiac Reprogramming: From Developmental Biology to Cardiac Regeneration (Circ Res. 2013;113:915–921) Perspectives for iPS Cell Technology: New Insights Into Human Physiology Involved in Somatic Mosaicism Engineering Adolescence: Maturation of Human Pluripotent Stem Cell-Derived Cardiomyocytes iPS Cells for Post-myocardial Infarction Repair: Remarkable Opportunities and Challenges Shinya Yamanaka, Guest Editor
Perspectives for Induced Pluripotent Stem Cell Technology New Insights Into Human Physiology Involved in Somatic Mosaicism Naoki Nagata, Shinya Yamanaka
Abstract: Induced pluripotent stem cell technology makes in vitro reprogramming of somatic cells from individuals with various genetic backgrounds possible. By applying this technology, it is possible to produce pluripotent stem cells from biopsy samples of arbitrarily selected individuals with various genetic backgrounds and to subsequently maintain, expand, and stock these cells. From these induced pluripotent stem cells, target cells and tissues can be generated after certain differentiation processes. These target cells/tissues are expected to be useful in regenerative medicine, disease modeling, drug screening, toxicology testing, and proof-of-concept studies in drug development. Therefore, the number of publications concerning induced pluripotent stem cells has recently been increasing rapidly, demonstrating that this technology has begun to infiltrate many aspects of stem cell biology and medical applications. In this review, we discuss the perspectives of induced pluripotent stem cell technology for modeling human diseases. In particular, we focus on the cloning event occurring through the reprogramming process and its ability to let us analyze the development of complex disease-harboring somatic mosaicism. (Circ Res. 2014;114:505-510.) Key Words: induced pluripotent stem cells
I
nduced pluripotent stem (iPS) cell technology involves the generation of pluripotent stem cells from adult somatic cells by the exogenous expression of specific reprogramming factors1,2 (see Box). This technology opened the door for us to access a robust platform demonstrating in vitro cellular reprogramming from certain somatic cells to pluripotent status. Recently, Cahan and Daley8 conducted a bibliometric analysis of the pluripotent stem cells (including iPS cells, embryonic stem [ES] cells, and embryonic carcinoma cells) generated since 2010. They found that the number of publications concerning ES cells has decreased, whereas the number of publications
■
mosaicism
concerning the applications of pluripotent stem cells has increased. They pointed out that it is the increase in the studies of iPS cells that brought about this change in the research trend, because it gave scientists access to pluripotent stem cells with robust and reproducible technology. As Cahan and Daley8 have shown, iPS cells quickly became recognized as an innovative research tool for the study of biology and medicine. In this review, we focus on iPS cell technology, with a special emphasis on its cloning event, which is essential for the reprogramming process, and discuss perspectives related to the creation of in vitro models of human physiological conditions, including complex diseases.
Original received November 15, 2013; revision received December 27, 2013; accepted January 6, 2014. In December 2013, the average time from submission to first decision for all original research papers submitted to Circulation Research was 11.66 days. From the Department of Reprogramming Science, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan (N.N., S.Y.); and Gladstone Institute of Cardiovascular Disease, San Francisco, CA (S.Y.). Correspondence to Shinya Yamanaka, MD, PhD, Center for iPS cell Research and Application, Kyoto University, Kyoto 606-8507, Japan. E-mail [email protected] © 2014 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.114.303043
Downloaded from http://circres.ahajournals.org/ 505at YORK UNIV SCOTT LIB on June 2, 2014
506 Circulation Research January 31, 2014
Nonstandard Abbreviations and Acronyms CINCA DS ES iPS
chronic infantile neurologic cutaneous and articular Down syndrome embryonic stem induced pluripotent stem
Discovery of Induced Pluripotent Stem Cells Induced pluripotent stem (iPS) cells are generated from mouse cells by the ectopic expression of key transcription factors (Oct3/4, Sox2, c-Myc, Klf4).1 The iPS cells were indistinguishable from embryonic stem cells in terms of their morphology, gene expression, self-renewal capacity, and pluripotency. Therefore, under this process, the somatic memory of the subject cells was reprogrammed to the stage of blastocyst inner cells. Further experiments confirmed the contribution of iPS cells to mammalian (mouse) ontogenesis. The mouse iPS cells have been confirmed to have a chimeric contribution following blastocyst injection and have also been proven to have competency for the germ line.3–5 Subsequently, human iPS cells were established by 3 laboratories at almost the same time using the same approaches.2,6,7 This shows that the iPS cell technology was already highly reproducible and accessible to several laboratories and that these cells can be readily used to perform experiments in molecular biology and mammalian cell culture. These iPS cells met the criteria proposed for the definition of human embryonic stem cells, with the exception that the iPS cells are not derived from embryos.
iPS Cells for In Vitro Modeling of Native Physiological Conditions Immortalized cells and primary cell lines have been the candidate cells used for creating human physiological models thus far. However, both of these types of cells are associated with serious defects when used for such purposes. Although immortalized cell lines can be maintained as homogenous cell populations at low cost, they lack important aspects of the native functions of cells. Likewise, although primary cell lines can provide fully differentiated cells that have a close approximation of the native function, the collection of all cell types is not possible because of the level of invasion required to collect them, and the limited proliferative activity of primary cells can make reproducing experiments difficult.9 On the contrary, human-derived iPS and ES cell lines can overcome these disadvantages. After the expansion of these pluripotent cell lines, a sufficient number of the desired types of cells can be prepared by using appropriate differentiation protocols, that is, these technologies can provide an opportunity to maintain specific somatic cells on a large scale and in a renewable way. However, there are still some limitations that need to be overcome. For example, it has been pointed out that cardiomyocytes differentiated from iPS cells using the current differentiation protocols yield relatively immature cells.10–12 Therefore, further improvement of the differentiation and
maturation protocols will be needed to more accurately recapitulate the phenotype in iPS cell–derived differentiated cells. Human ES cells carrying mutation for some genetic disorders have been reported (eg, Huntington disease, cystic fibrosis, myotonic dystrophy type 1, and Fragile X syndrome).13,14 However, there are certain ethical concerns associated with developing ES cells obtained from in vitro fertilization clinics for the study of genetic diseases.15 In contrast, such ethical issues are overcome with the use of disease-specific iPS cell lines, because they can be generated from somatic cells such as fibroblasts or peripheral blood cells, which can be collected by minimally invasive procedures and do not require the destruction of a human embryo.
iPS Cells From Arbitrarily Selected Individuals Another advantage of using iPS cells in medical research is that this technology makes it possible to obtain human pluripotent stem cells from arbitrarily selected individuals with various genetic backgrounds, including patients with various diseases. In 2008, the first patient-specific iPS cells were generated.16,17 Since then, abundant studies of disease models that represent the human condition with high fidelity have been reported,18,19 particularly those aiming to investigate diseases that are caused by a single gene mutation. In most of the studies, iPS cell lines from unaffected or healthy donors were used as controls. However, recent genome-wide association studies using a set of controls derived from different individuals showed significant experimental noise attributable to genomic variations. Moreover, these controls cannot really be defined as healthy, because each person carries 50 to 100 diseaseassociated genetic variations.20 One way to address these issues is to obtain isogenic controls by correcting mutated genes in patient-specific iPS cell lines.21–27 The recent progresses in genetic engineering technologies, such as Transcription Activator-Like Effector Nuclease (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs), will facilitate this strategy.27,28 Somatic mutations can give rise to a broad range of diseases, including cancer, and noncancerous mosaic diseases.18 Recently, heterogeneous patient-specific iPS cell lines have been generated from the somatic cells of patients with various mosaic diseases, such as Down syndrome (DS),29–35 Fragile X syndrome,36 Rett syndrome,37–42 Fanconi anemia,43 and chronic infantile neurologic cutaneous and articular (CINCA) syndrome.44 Even though there are some limitations associated with the kinds of diseases that can be modeled, using iPS cells derived from patients with somatic mosaicism has an advantage in that it allows analyses with isogenic controls. In these cases, both mutant and wild-type iPS cell clones can be generated from the same patients. Each pair of clones theoretically has the same genetic background, except for the altered chromosome or mutated gene(s) and should serve as an ideal pair of mutant and isogenic control clones, thereby allowing for highly reliable studies of the impact of the mutation. It should be mentioned that we could not completely exclude the introduction of new mutations during the induction of iPS cells and subsequent cultivation. The recently improved next-generation DNA sequencing and bioinformatics techniques should be useful in detecting such acquired mutations and increasing the quality of experiments.
Downloaded from http://circres.ahajournals.org/ at YORK UNIV SCOTT LIB on June 2, 2014
Nagata and Yamanaka Perspectives for iPS Cell Technology 507 The following examples of patient-specific iPS cells generated from mosaic cases of DS and CINCA syndrome illustrate the advantage of using iPS cell technology for disease modeling.
Somatic Mosaicism in DS DS is the most frequent form of mental retardation and is caused by autosomal trisomy of all or a critical portion of chromosome 21. Patients with DS are reported to present with multiple disorders (eg, congenital heart defects, particularly atrioventricular septal defect, leukemia, and early- onset Alzheimer disease [OMIM 190685]).45 A cytogenetic study showed that, in the United States, most infants with DS (≈95%) had full trisomy 21; in ≈3% of patients, one copy was translocated to another acrocentric chromosome, and in ≈2% of cases, live-born trisomy 21 individuals were recognized to have mosaicism for a trisomic cell line.46 Mosaicism results from the abnormal division of some cells after fertilization, with some of the cells having 47 chromosomes and the others being normal. In each of these conditions, the outcome of this disorder is determined by the particular chromosome being duplicated, as well as by the proportion of cells in the body carrying the abnormality.18 Papavassiliou et al47 observed a correlation between the frequency of trisomic cells and the patient phenotype. They detected a significant inverse correlation between the frequency of trisomic cells and IQ scores in individuals with mosaic trisomy 21. Mosaic trisomies have also been reported on other chromosomes, such as chromosomes 1, 8, 9, 13, 16, 17, 18, and 22 (as reviewed by Poduri et al18). Many attempts have been made to model DS to investigate the detailed pathophysiology of the condition. Generally, biopsy samples of brains are not available from patients, and thus, mouse models are currently the primary tools used to study the pathogenesis of DS. However, brain development differs in mice and humans.48 Although some human ES cells with trisomy 21 have been generated,49 the establishment of human ES cells from patients with DS remains ethically challenging.
Capturing Trisomic and Disomic Cells From Mosaic Patients Using the iPS Cell Technology: DS as an Example DS/trisomy 21 patient-specific iPS cell lines have been generated from various cases, including mosaic patients.17,29–35
DS Patient-derived iPS cell lines
DS-Patient / Mosaicism Biopsy sample
Reprogramming
Cloning
Disomic cell
Trisomic cell Transcription factors
Figure. Generation of induced pluripotent stem (iPS) cell lines from patient with mosaic Down syndrome (DS).
Recently, 2 groups reported the generation of iPS cell lines from fibroblasts that were derived from patients with DS with mosaicism.34,48 They confirmed that the trisomy 21 karyotype was present in the DS iPS cells; however, they also captured iPS cell lines from the patient’s fibroblasts, which were found to possess a disomic karyotype. This seemed to be the result of the reprogramming of euploid cells from the mosaic donor’s fibroblast population. DNA profiling of the corresponding cell lines revealed that these disomic and trisomic cell lines actually came from the same donor. The patient’s disomic cell line provides an ideal experimental control, because it can be used cancel the noise in the genetic background that is often observed using so-called “healthy” individuals as controls. This system presents a powerful method for analyzing multiple genotype–phenotype associations in complex diseases that harbor multiple candidate mutations, such as chromosomal aberrations (Figure).
Somatic Mosaicism in CINCA Syndrome Beside DS, patient-specific iPS cell lines have also been generated from the somatic cells of patients with autosomal mosaic point mutations. For example, CINCA syndrome44 is a dominantly inherited autoinflammatory disease characterized by cutaneous symptoms, central nervous system involvement, and arthropathy (OMIM 607115). Saito et al50 described the first case of a patient with somatic mosaicism in their NLRP3 (also known as CIAS1) gene mutation. It has been observed that 30% to 40% of all patients have mutations in NLRP3 in only a small number (≈10%) of somatic cells,50,51 despite the fact that nearly half of all patients with CINCA syndrome carry heterozygous gain-of-function mutations of the gene.9,52 Recently, Tanaka et al44 generated both NLP3 mutant and nonmutant iPS cell lines from patients with CINCA syndrome with somatic mosaicism and described their differentiation into macrophages. In this case, they succeeded in recapitulating the disease-relevant phenotype using only mutant macrophages derived from iPS cells, demonstrating that NLRP3-mutant macrophages are responsible for the pathogenesis of mosaic CINCA syndrome. Moreover, they succeeded in developing a drug screening system with the macrophages derived from iPS cells and illustrated the usefulness of iPS cell technology as a platform for drug discovery.
A New Approach to Studying Somatic Mutations Using the iPS Cell Technology Recently, somatic mosaicism has been reported in a variety of tissues from healthy individuals, suggesting that it has physiological functions.29 In such cases, iPS cell technology can provide a universally applicable strategy to capture these somatic mosaicisms in human populations, allowing their impact to be evaluated. Evidence from recent studies using single-nucleotide polymorphism microarrays or the next-generation sequencing technology demonstrates that a certain proportion of mutations that are detected in iPS cells come from the heterogeneous donor cell population, attributable to somatic mosaicism.53–55 The cells that carry pre-existing genetic alterations, such as singlenucleotide variants and copy number variations, are captured by the cloning event through the reprogramming process.
Downloaded from http://circres.ahajournals.org/ at YORK UNIV SCOTT LIB on June 2, 2014
508 Circulation Research January 31, 2014 These single-nucleotide variants and copy number variations in a minor population of somatic cells have to date been overlooked because of the difficulty in detecting them. However, applying iPS cell technology allows for improved detection of such low-level mosaicism.
environmental/aging association successfully at the molecular level by applying iPS cell technology. Moreover, iPS cell technology holds the potential to lead to new insights into the human physiology associated with somatic mosaicism.
Somatic Mosaicisms in Cardiovascular Diseases
We thank Drs Yoshinori Yoshida and Kazutoshi Takahashi for their valuable scientific discussions. We also thank Rie Kato and Yoko Miyake for their valuable administrative support.
Systematic analyses of Leipzig heart collections have shown somatic mutations in several transcription factor genes (NKX2-5, TBX5, GATA4, and HEY2) associated with complex congenital heart diseases, including atrial septal, ventricular, and atrioventricular septal defects.56–61 In these cases, each mutation was detected in the affected heart tissues, but not in the normal heart tissues, of the same patients. In this situation, mutant cardiomyocytes can be provided by gene editing of wild-type iPS cell lines. Another set of examples of mosaicism was found in somatic mutations associated with arrhythmia.62–64 Mosaicism in connexin 40 and connexin 43 is associated with atrial fibrillation.63,64 Interestingly, subcloning analysis estimated allelic frequencies of mutant alleles at 20% to 34% within the patient’s cardiac tissue specimens; however, these mutations were not detected in lymphocytes. Each mutant connexin- expressing cell was observed loss of contribution to gap junction formations, as well as electrophysiological functions. To further investigate the pathophysiology of those atrial fibrillations with somatic mosaicism of connexins, modeling of in vitro cardiac syncytia in which each cardiomyocyte is individually expressing either wild-type or mutant connexins provides a powerful tool. Recently, Kadota et al65 created cardiomyocyte sheets from human iPS/ES cells. They demonstrated that these sheets are capable of generating re-entrant arrhythmia models when stimulated with high-frequency electric pulses and subsequently showed their usefulness for screening and testing drugs with antiarrhythmic potential. Although further technological improvements are needed, this approach can provide a more precise in vitro micro–re-entrant model for the mosaic cardiac syncytium. In this case, we may choose to obtain mutated cardiac cells by gene-editing technology based on unaffected/well-characterized iPS cells because to date mutant connexin 40–expressing and connexin 43–expressing cells have only been detected in patient cardiac tissues, which are difficult to obtain by biopsy.
Concluding Remarks Somatic variants are known to be potentially responsible for various diseases. However, the extent of somatic variation may have been markedly underestimated.66 As we discussed above, iPS cell technology allows us to approach human biology with higher resolution at the cellular level through the cell cloning process, as part of the reprogramming process. This approach holds great promise for studies on the native human physiological processes, as well as the development and pathology of human diseases. For example, in patients with trisomy 21, acquired mosaicism was observed in adult patients.67 Although there are still several technical hurdles that have to be overcome, we may be able to analyze such a disease/
Acknowledgments
Sources of Funding This work was supported by a grant from the Funding Program for World-Leading Innovative Research and Development on Science and Technology (FIRST Program) of the Japanese Society for the Promotion of Science (JSPS) and by a grant from Research Center Network for Realization of Regenerative Medicine of the Japan Science and Technology Agency (JST).
Disclosures S. Yamanaka is a member without salary of the scientific advisory boards of iPierian, iPS Academia Japan, Megakaryon Corporation, and HEALIOS K. K. Japan. The other author reports no conflicts.
References 1. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663–676. 2. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131:861–872. 3. Maherali N, Sridharan R, Xie W, Utikal J, Eminli S, Arnold K, Stadtfeld M, Yachechko R, Tchieu J, Jaenisch R, Plath K, Hochedlinger K. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell. 2007;1:55–70. 4. Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature. 2007;448:313–317. 5. Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, Hochedlinger K, Bernstein BE, Jaenisch R. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature. 2007;448:318–324. 6. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007;318:1917–1920. 7. Park IH, Zhao R, West JA, Yabuuchi A, Huo H, Ince TA, Lerou PH, Lensch MW, Daley GQ. Reprogramming of human somatic cells to pluripotency with defined factors. Nature. 2008;451:141–146. 8. Cahan P, Daley GQ. Origins and implications of pluripotent stem cell variability and heterogeneity. Nat Rev Mol Cell Biol. 2013;14:357–368. 9. Ebert AD, Shelley BC, Hurley AM, Onorati M, Castiglioni V, Patitucci TN, Svendsen SP, Mattis VB, McGivern JV, Schwab AJ, Sareen D, Kim HW, Cattaneo E, Svendsen CN. EZ spheres: a stable and expandable culture system for the generation of pre-rosette multipotent stem cells from human ESCs and iPSCs. Stem Cell Res. 2013;10:417–427. 10. Jung CB, Moretti A, Mederos y Schnitzler M, et al. Dantrolene rescues arrhythmogenic RYR2 defect in a patient-specific stem cell model of catecholaminergic polymorphic ventricular tachycardia. EMBO Mol Med. 2012;4:180–191. 11. Dirschinger RJ, Goedel A, Moretti A, Laugwitz KL, Sinnecker D. Recapitulating long-QT syndrome using induced pluripotent stem cell technology. Pediatr Cardiol. 2012;33:950–958. 12. Friedrichs S, Malan D, Sasse P. Modeling long QT syndromes using induced pluripotent stem cells: current progress and future challenges. Trends Cardiovasc Med. 2013;23:91–98. 13. Mateizel I, De Temmerman N, Ullmann U, Cauffman G, Sermon K, Van de Velde H, De Rycke M, Degreef E, Devroey P, Liebaers I, Van Steirteghem A. Derivation of human embryonic stem cell lines from embryos obtained after IVF and after PGD for monogenic disorders. Hum Reprod. 2006;21:503–511. 14. Eiges R, Urbach A, Malcov M, Frumkin T, Schwartz T, Amit A, Yaron Y, Eden A, Yanuka O, Benvenisty N, Ben-Yosef D. Developmental
Downloaded from http://circres.ahajournals.org/ at YORK UNIV SCOTT LIB on June 2, 2014
Nagata and Yamanaka Perspectives for iPS Cell Technology 509 study of fragile X syndrome using human embryonic stem cells derived from preimplantation genetically diagnosed embryos. Cell Stem Cell. 2007;1:568–577. 15. Pomp O, Colman A. Disease modelling using induced pluripotent stem cells: status and prospects. Bioessays. 2013;35:271–280. 16. Dimos JT, Rodolfa KT, Niakan KK, Weisenthal LM, Mitsumoto H, Chung W, Croft GF, Saphier G, Leibel R, Goland R, Wichterle H, Henderson CE, Eggan K. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science. 2008;321:1218–1221. 17. Park IH, Arora N, Huo H, Maherali N, Ahfeldt T, Shimamura A, Lensch MW, Cowan C, Hochedlinger K, Daley GQ. Disease-specific induced pluripotent stem cells. Cell. 2008;134:877–886. 18. Poduri A, Evrony GD, Cai X, Walsh CA. Somatic mutation, genomic variation, and neurological disease. Science. 2013;341:1237758. 19. Trounson A, Shepard KA, DeWitt ND. Human disease modeling with induced pluripotent stem cells. Curr Opin Genet Dev. 2012;22:509–516. 20. Abecasis GR, Altshuler D, Auton A, Brooks LD, Durbin RM, Gibbs RA, Hurles ME, McVean GA; 1000 Genomes Project Consortium. A map of human genome variation from population-scale sequencing. Nature. 2010;467:1061–1073. 21. Kazuki Y, Hiratsuka M, Takiguchi M, et al. Complete genetic cor rection of ips cells from Duchenne muscular dystrophy. Mol Ther. 2010;18:386–393. 22. Howden SE, Gore A, Li Z, et al. Genetic correction and analysis of induced pluripotent stem cells from a patient with gyrate atrophy. Proc Natl Acad Sci U S A. 2011;108:6537–6542. 23. Liu GH, Suzuki K, Qu J, et al. Targeted gene correction of laminopathyassociated LMNA mutations in patient-specific iPSCs. Cell Stem Cell. 2011;8:688–694. 24. Tolar J, Park IH, Xia L, Lees CJ, Peacock B, Webber B, McElmurry RT, Eide CR, Orchard PJ, Kyba M, Osborn MJ, Lund TC, Wagner JE, Daley GQ, Blazar BR. Hematopoietic differentiation of induced pluripotent stem cells from patients with mucopolysaccharidosis type I (Hurler syndrome). Blood. 2011;117:839–847. 25. Bellin M, Casini S, Davis RP, D’Aniello C, Haas J, Ward-van Oostwaard D, Tertoolen LG, Jung CB, Elliott DA, Welling A, Laugwitz KL, Moretti A, Mummery CL. Isogenic human pluripotent stem cell pairs reveal the role of a KCNH2 mutation in long-QT syndrome. EMBO J. 2013;32:3161–3175. 26. Fattahi F, Asgari S, Pournasr B, Seifinejad A, Totonchi M, Taei A, Aghdami N, Salekdeh GH, Baharvand H. Disease-corrected hepatocytelike cells from familial hypercholesterolemia-induced pluripotent stem cells. Mol Biotechnol. 2013;54:863–873. 27. Yusa K, Rashid ST, Strick-Marchand H, et al. Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells. Nature. 2011;478:391–394. 28. Merkle FT, Eggan K. Modeling human disease with pluripotent stem cells: from genome association to function. Cell Stem Cell. 2013;12:656–668. 29. Shi Y, Kirwan P, Smith J, MacLean G, Orkin SH, Livesey FJ. A human stem cell model of early Alzheimer’s disease pathology in Down syndrome. Sci Transl Med. 2012;4:124ra29. 30. Mou X, Wu Y, Cao H, Meng Q, Wang Q, Sun C, Hu S, Ma Y, Zhang H. Generation of disease-specific induced pluripotent stem cells from patients with different karyotypes of Down syndrome. Stem Cell Res Ther. 2012;3:14. 31. Li LB, Chang KH, Wang PR, Hirata RK, Papayannopoulou T, Russell DW. Trisomy correction in Down syndrome induced pluripotent stem cells. Cell Stem Cell. 2012;11:615–619. 32. Biesecker LG, Spinner NB. A genomic view of mosaicism and human disease. Nat Rev Genet. 2013;14:307–320. 33. Maclean GA, Menne TF, Guo G, Sanchez DJ, Park IH, Daley GQ, Orkin SH. Altered hematopoiesis in trisomy 21 as revealed through in vitro differentiation of isogenic human pluripotent cells. Proc Natl Acad Sci U S A. 2012;109:17567–17572. 34. Weick JP, Held DL, Bonadurer GF 3rd, et al. Deficits in human trisomy 21 iPSCs and neurons. Proc Natl Acad Sci U S A. 2013;110:9962–9967. 35. Lu HE, Yang YC, Chen SM, Su HL, Huang PC, Tsai MS, Wang TH, Tseng CP, Hwang SM. Modeling neurogenesis impairment in Down syndrome with induced pluripotent stem cells from Trisomy 21 amniotic fluid cells. Exp Cell Res. 2013;319:498–505. 36. Sheridan SD, Theriault KM, Reis SA, Zhou F, Madison JM, Daheron L, Loring JF, Haggarty SJ. Epigenetic characterization of the FMR1 gene and aberrant neurodevelopment in human induced pluripotent stem cell models of fragile X syndrome. PLoS One. 2011;6:e26203.
37. Marchetto MC, Carromeu C, Acab A, Yu D, Yeo GW, Mu Y, Chen G, Gage FH, Muotri AR. A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell. 2010;143:527–539. 38. Amenduni M, De Filippis R, Cheung AY, Disciglio V, Epistolato MC, Ariani F, Mari F, Mencarelli MA, Hayek Y, Renieri A, Ellis J, Meloni I. iPS cells to model CDKL5-related disorders. Eur J Hum Genet. 2011;19:1246–1255. 39. Ananiev G, Williams EC, Li H, Chang Q. Isogenic pairs of wild type and mutant induced pluripotent stem cell (iPSC) lines from Rett syndrome patients as in vitro disease model. PLoS One. 2011;6:e25255. 40. Cheung AY, Horvath LM, Grafodatskaya D, Pasceri P, Weksberg R, Hotta A, Carrel L, Ellis J. Isolation of MECP2-null Rett Syndrome patient hiPS cells and isogenic controls through X-chromosome inactivation. Hum Mol Genet. 2011;20:2103–2115. 41. Kim KY, Hysolli E, Park IH. Neuronal maturation defect in induced pluripotent stem cells from patients with Rett syndrome. Proc Natl Acad Sci U S A. 2011;108:14169–14174. 42. Pomp O, Dreesen O, Leong DF, Meller-Pomp O, Tan TT, Zhou F, Colman A. Unexpected X chromosome skewing during culture and reprogramming of human somatic cells can be alleviated by exogenous telomerase. Cell Stem Cell. 2011;9:156–165. 43. Müller LU, Schlaeger TM, DeVine AL, Williams DA. Induced pluripotent stem cells as a tool for gaining new insights into Fanconi anemia. Cell Cycle. 2012;11:2985–2990. 44. Tanaka T, Takahashi K, Yamane M, et al. Induced pluripotent stem cells from CINCA syndrome patients as a model for dissecting somatic mosaicism and drug discovery. Blood. 2012;120:1299–1308. 45. Mégarbané A, Ravel A, Mircher C, Sturtz F, Grattau Y, Rethoré MO, Delabar JM, Mobley WC. The 50th anniversary of the discovery of trisomy 21: the past, present, and future of research and treatment of Down syndrome. Genet Med. 2009;11:611–616. 46. Shin M, Siffel C, Correa A. Survival of children with mosaic Down syndrome. Am J Med Genet A. 2010;152A:800–801. 47. Papavassiliou P, York TP, Gursoy N, Hill G, Nicely LV, Sundaram U, McClain A, Aggen SH, Eaves L, Riley B, Jackson-Cook C. The phenotype of persons having mosaicism for trisomy 21/Down syndrome reflects the percentage of trisomic cells present in different tissues. Am J Med Genet A. 2009;149A:573–583. 48. Briggs JA, Sun J, Shepherd J, Ovchinnikov DA, Chung TL, Nayler SP, Kao LP, Morrow CA, Thakar NY, Soo SY, Peura T, Grimmond S, Wolvetang EJ. Integration-free induced pluripotent stem cells model genetic and neural developmental features of down syndrome etiology. Stem Cells. 2013;31:467–478. 49. Biancotti JC, Narwani K, Buehler N, Mandefro B, Golan-Lev T, Yanuka O, Clark A, Hill D, Benvenisty N, Lavon N. Human embryonic stem cells as models for aneuploid chromosomal syndromes. Stem Cells. 2010;28:1530–1540. 50. Saito M, Fujisawa A, Nishikomori R, Kambe N, Nakata-Hizume M, Yoshimoto M, Ohmori K, Okafuji I, Yoshioka T, Kusunoki T, Miyachi Y, Heike T, Nakahata T. Somatic mosaicism of CIAS1 in a patient with chronic infantile neurologic, cutaneous, articular syndrome. Arthritis Rheum. 2005;52:3579–3585. 51. Tanaka N, Izawa K, Saito MK, et al. High incidence of NLRP3 somatic mosaicism in patients with chronic infantile neurologic, cutaneous, articular syndrome: results of an International Multicenter Collaborative Study. Arthritis Rheum. 2011;63:3625–3632. 52. Feldmann J, Prieur AM, Quartier P, Berquin P, Certain S, Cortis E, TeillacHamel D, Fischer A, de Saint Basile G. Chronic infantile neurological cutaneous and articular syndrome is caused by mutations in CIAS1, a gene highly expressed in polymorphonuclear cells and chondrocytes. Am J Hum Genet. 2002;71:198–203. 53. Abyzov A, Mariani J, Palejev D, et al. Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells. Nature. 2012;492:438–442. 54. Young MA, Larson DE, Sun CW, et al. Background mutations in parental cells account for most of the genetic heterogeneity of induced pluripotent stem cells. Cell Stem Cell. 2012;10:570–582. 55. Chou ST, Byrska-Bishop M, Tober JM, Yao Y, Vandorn D, Opalinska JB, Mills JA, Choi JK, Speck NA, Gadue P, Hardison RC, Nemiroff RL, French DL, Weiss MJ. Trisomy 21-associated defects in human primitive hematopoiesis revealed through induced pluripotent stem cells. Proc Natl Acad Sci U S A. 2012;109:17573–17578. 56. Reamon-Buettner SM, Borlak J. Somatic NKX2-5 mutations as a novel mechanism of disease in complex congenital heart disease. J Med Genet. 2004;41:684–690.
Downloaded from http://circres.ahajournals.org/ at YORK UNIV SCOTT LIB on June 2, 2014
510 Circulation Research January 31, 2014 57. Reamon-Buettner SM, Hecker H, Spanel-Borowski K, Craatz S, Kuenzel E, Borlak J. Novel NKX2-5 mutations in diseased heart tissues of patients with cardiac malformations. Am J Pathol. 2004;164: 2117–2125. 58. Reamon-Buettner SM, Borlak J. TBX5 mutations in non-Holt-Oram syndrome (HOS) malformed hearts. Hum Mutat. 2004;24:104. 59. Reamon-Buettner SM, Borlak J. GATA4 zinc finger mutations as a molecular rationale for septation defects of the human heart. J Med Genet. 2005;42:e32. 60. Reamon-Buettner SM, Borlak J. HEY2 mutations in malformed hearts. Hum Mutat. 2006;27:118. 61. Weismann CG, Gelb BD. The genetics of congenital heart disease: a review of recent developments. Curr Opin Cardiol. 2007;22:200–206. 62. Lerman BB, Dong B, Stein KM, Markowitz SM, Linden J, Catanzaro DF. Right ventricular outflow tract tachycardia due to a somatic cell mutation in G protein subunitalphai2. J Clin Invest. 1998;101:2862–2868.
63. Gollob MH, Jones DL, Krahn AD, et al. Somatic mutations in the connexin 40 gene (GJA5) in atrial fibrillation. N Engl J Med. 2006;354:2677–2688. 64. Thibodeau IL, Xu J, Li Q, Liu G, Lam K, Veinot JP, Birnie DH, Jones DL, Krahn AD, Lemery R, Nicholson BJ, Gollob MH. Paradigm of genetic mosaicism and lone atrial fibrillation: physiological characterization of a connexin 43-deletion mutant identified from atrial tissue. Circulation. 2010;122:236–244. 65. Kadota S, Minami I, Morone N, Heuser JE, Agladze K, Nakatsuji N. Development of a reentrant arrhythmia model in human pluripotent stem cell-derived cardiac cell sheets. Eur Heart J. 2013;34:1147–1156. 66. Erickson RP. Somatic gene mutation and human disease other than cancer: an update. Mutat Res. 2010;705:96–106. 67. Jenkins EC, Schupf N, Genovese M, Ye LL, Kapell D, Canto B, Harris M, Devenny D, Lee JH, Brown WT. Increased low-level chromosome 21 mosaicism in older individuals with Down syndrome. Am J Med Genet. 1997;68:147–151.
Downloaded from http://circres.ahajournals.org/ at YORK UNIV SCOTT LIB on June 2, 2014
